Glucose metabolism modulating compounds

ABSTRACT

The present invention provides, inter alia, dihydropyridone compounds and compositions, including analogs of a vesicular monoamine transporter type 2 (VMAT2) antagonist. The present invention also provides methods of using such compounds/analogs for modulating glucose levels, and/or preventing, treating, or ameliorating the effects of diabetes and hyperglycemia.

RELATED APPLICATIONS

This application is related to and claims priority from U.S. patentapplication Ser. No. 61/123,785, filed Apr. 11, 2008, the content ofwhich is incorporated by reference herein in its entirety.

GOVERNMENT FUNDING

This invention was made with government support under 5 R01 DK 63567,1R21-DK70192-01, and 2 RO1 DK63567-05 awarded by the National Institutesof Health. The government has certain rights in the invention.

FIELD OF THE INVENTION

The present invention relates, inter alia, to compounds, pharmaceuticalcompositions, and methods to modulate glucose metabolism and to prevent,treat, or ameliorate the effects of diabetes and hyperglycemia by, e.g.,interacting with VMAT2.

BACKGROUND OF THE INVENTION

Diabetes mellitus is a growing epidemic affecting hundreds of millionsof people worldwide. (Zimmer et al., 2001). Despite a recent explosionof new classes of hypoglycemic agents, the medical need remains largelyunmet and innovative diagnostics and therapeutics are still urgentlyneeded.

D-Glucose, with the synergistic effects of certain amino acids, is themajor physiological stimulus for insulin secretion (reviewed in (Henquin2000)). Net insulin production and glucose homeostasis, however, isregulated by a number of other molecules, including several classicalneurotransmitters (Ahren 2000; Brunicardi, et al. 1995) that actdirectly on beta cells, and indirectly through other target tissues suchas liver and skeletal muscle. Many of these molecules function asamplifying agents that have little or no effect by themselves, butenhance the signals generated by the beta cell glucose sensing apparatus(Henquin 2000). For example, during the cephalic phase of insulinrelease, acetylcholine (ACh) is released via islet parasympatheticinnervation. Beta cells express the M3 muscarinic receptor (Duttaroy, etal. 2004) and respond to exogenous ACh with increased inositol phosphateproduction, which in turn facilitates sodium (Na⁺) ion exit and calciumion entry. This results in augmented insulin vesicle exocytosis (Barker,et al. 2002).

The amino acid glutamate, the major excitatory neurotransmitter in thecentral nervous system, is present in both alpha- and beta-cells of theendocrine pancreas. Glutamate is stored in glucagon-containing granules(Hayashi, et al. 2003), and is proposed to enhance insulin secretionwhen it is released into the vicinity of islet cells (Storto, et al.2006). The presence of metabotropic glutamate receptors on alpha- andbeta-cells themselves suggests the presence of both autocrine andparacrine circuits within islet tissue involved in the regulation ofinsulin secretion (Brice, et al. 2002).

Other neurotransmitters, such as the monoamines, epinephrine andnorepinephine, acting both systemically and via nerve terminals in thevicinity of islets, may act to suppress glucose stimulated insulinsecretion by direct interaction with adrenoreceptors expressed (mainlythe alpha 2 receptor) on pancreatic beta cells (Ahren 2000; El-Mansouryand Morgan 1998). Beta cells of the endocrine pancreas also expressdopamine receptors (D2) and respond to exogenous dopamine with inhibitedglucose-stimulated insulin secretion (Ahren and Lundquist 1985;Niswender, et al. 2005; Rubi, et al. 2005; Shankar, et al. 2006).Purified islet tissue is a source of monoamines, and has been shown tocontain 5-hydroxytryptamine, epinephrine, norepinephrine and dopamine(Cegrell 1968; Ekholm, et al. 1971; Hansen and Hedeskov 1977; Lundquist,et al. 1989; Niswender et al. 2005; Wilson, et al. 1974).

Beta cells also have the biosynthetic apparatus to create, dispose of,and store specific neurotransmitters. For example, tyrosine hydroxylase,the enzyme responsible for catalyzing the conversion of L-tyrosine toL-3, 4-dihydroxyphenylalanine (L-DOPA), a precursor of dopamine, L-DOPAdecarboxylase, responsible for converting L-DOPA to dopamine (Rubi etal. 2005) and dopamine beta hydroxylase, the enzyme that catalyzes theconversion of dopamine to norepinephrine, are present in islet tissue(Borelli, et al. 2003; Iturriza and Thibault 1993). Thus, L-DOPA israpidly converted in islet beta-cells to dopamine (Ahren, et al. 1981;Borelli, et al. 1997).

Monoamine oxidase (MAO) is a catabolic enzyme responsible for theoxidative de-amination of monoamines, such as dopamine andcatecholamines, and maintains the cellular homeostasis of monoamines.The possible role of MAO in islet function has been studied, (Adeghateand Donath 1991) and MAO has been detected in both alpha- and beta-cellsof pancreatic islet cells, including beta cells (Feldman and Chapman1975a, b). Interestingly, some MAO inhibitors have been shown toantagonize glucose-induced insulin secretion (Aleyassine and Gardiner1975). The secretory granules of pancreatic beta cells store substantialamounts of calcium, dopamine and serotonin (Ahren and Lundquist 1985).

In the central nervous system, the storage of monoamineneurotransmitters in secretory organelles is mediated by a vesicularamine transporter. These molecules are expressed as integral membraneproteins of the lipid bilayer of secretory vesicles in neuronal andendocrine cells. An electrochemical gradient provides energy for thevesicular packaging of monoamines, such as dopamine, for later dischargeinto the synaptic space (reviewed by (Eiden, et al. 2004)). Bothimmunohistochemistry and gene expression studies show that islet tissueand the beta cells of the endocrine pancreas selectively express onlyone member of the family of vesicular amine transporters, vesicularmonoamine transporter type 2 (VMAT2) (Anlauf, et al. 2003).

VMAT2 is one member of the vesicular transporter family responsible forthe uptake and secretion of monoamine neurotransmitters in neurons andendocrine cells. (Zheng et al, 2006) Recent studies have shown thefeasibility of noninvasive measurements of the amount of VMAT2 in thepancreas as a useful biomarker of beta cell mass both in humans (R.Goland, et al. 2009) and rodents (Souza, et al. 2006) using[¹¹C]dihydrotetrabenazine (DTBZ) and positron emission tomography, butthe possible functional role of VMAT2, as expressed in islet tissue andbeta cells, in glucose metabolism has not yet been explored.

As indicated, endogenously synthesized and/or stored monoamineneurotransmitters appear to participate in paracrine regulation ofinsulin secretion and entrainment of the activity of various cellswithin islets (Borelli and Gagliardino 2001). Given the important roleof vesicular amine transporters in the storage and distribution ofmonoamine neurotransmitters, the possible of role of VMAT2 inglucose-stimulated insulin secretion was explored using theVMAT2-specific antagonist, tetrabenazine (TBZ) (Scherman, et al. 1983).TBZ acts as a reversible inhibitor of monoamine uptake into granularvesicles of presynaptic neurons (Pettibone, et al. 1984) through itsability to bind to VMAT2 (Scherman 1986) thereby facilitating monoaminedegradation by MAO. Monoamine neurotransmitters that are depleted viaVMAT2 inhibition by TBZ include serotonin, dopamine, and norepinephrine.Administration of TBZ to rats (plasma elimination with half life,t_(1/2) equals 2 hours) reduces dopamine levels by 40%, serotonin by44%, and norepinephrine by 41% in the brain (Lane, et al. 1976).

Although there are other vesicular amine transporters (e.g. vesicularmonoamine transporter type 1, or VMAT1), tetrabenazine is highlyspecific for VMAT2, binds to the transporter with a dissociationconstant in the nanomolar range, and displays a more than 10,000-foldreduced affinity towards VMAT1 (Erickson, et al. 1996; Varoqui andErickson, 1997). Given the known effects of monoamine neurotransmitterson insulin secretion, the expression of VMAT2 by beta cells and theantagonist action of TBZ on monoamine transport, it would be desirableto identify compounds, pharmaceutical compositions and methods tomodulate glucose metabolism and insulin secretion to, e.g., prevent,treat, or ameliorate the effects of impaired glucose metabolism, suchas, e.g., diabetes and hyperglycemia.

SUMMARY OF THE INVENTION

Thus, one object of the invention is to show that VMAT2 expressed inbeta cells of the endocrine pancreas plays a role in the regulation ofinsulin production and glucose homeostasis in vivo. Another object ofthe invention is to show that the glucose tolerance enhancing effects ofTBZ is mediated by the depletion of dopamine following the antagonism ofVMAT2. A further object of the invention is to show that certaincompounds modulate blood glucose levels. These and other objects of theinvention are disclosed in more detail in the embodiments that follow.

One embodiment of the present invention is a compound of formula I:

-   -   wherein R₁, R₂, R₃, R₆, and R₇ are independently selected from        H, halogen, hydroxyl, C₁₋₈alkyl, C₁-8alkenyl, C₁₋₈alkynyl,        C₁₋₈alkoxy, 3- to 8-membered carbocyclic or heterocyclic, aryl,        heteroaryl, C₁₋₄aralkyl, residues of glycolic acid, ethylene        glycol/propylene glycol copolymers, carboxylate, ester, amide,        carbohydrate, amino acid, alditol, OC(X)₂COOH, SC(X)₂COOH,        NHCHXCOOH, COY, CO₂Y, sulfate, sulfonamide, sulfoxide,        sulfonate, sulfone, thioalkyl, thioester, propylphthalimide, and        thioether;    -   R₄ and R₅, which are attached to one or more positions of at        least one carbon atom of the respective rings, are independently        selected from H, halogen, hydroxyl, C₁₋₈alkyl, C₁₋₈alkenyl,        C₁₋₈alkynyl, C₁₋₈alkoxy, 3- to 8-membered carbocyclic or        heterocyclic, aryl, heteroaryl, C₁₋₄aralkyl, residues of        glycolic acid, ethylene glycol/propylene glycol copolymers,        carboxylate, ester, amide, carbohydrate, amino acid, alditol,        OC(X)₂COOH, SC(X)₂COOH, NHCHXCOOH, COY, CO₂Y, sulfate,        sulfonamide, sulfoxide, sulfonate, sulfone, thioalkyl,        thioester, propylphthalimide, and thioether;    -   X is selected from H, C₁₋₈alkyl, C₁₋₈alkenyl, C₁₋₈alkynyl,        carbocycle, aryl, heteroaryl, heterocycle, alkylaryl,        alkylheteroaryl, and alkylheterocycle, wherein each alkyl,        carbocycle, aryl, heteroaryl, heterocycle, alkylaryl,        alkylheteroaryl, and alkylheterocycle may be optionally        substituted with at least one substituent;    -   Y is selected from H, C₁₋₈alkyl, C₁₋₈alkenyl, C₁₋₈alkynyl, aryl,        carbocycle, heteroaryl, heterocycle, alkylaryl, alkylheteroaryl,        alkylheterocycle, and heteroaromatic, wherein each alkyl,        alkenyl, alkynyl, aryl, carbocycle, heteroaryl, heterocycle,        alkylaryl, alkylheteroaryl, alkylheterocycle, and heteroaromatic        may be optionally substituted with at least one substituent; and    -   --- is an optional bond, wherein the optional bond is a single        bond or a double bond;        or an enantiomer, optical isomer, diastereomer, N-oxide,        crystalline form, hydrate, or pharmaceutically acceptable salt        thereof.

Another embodiment of the present invention is a compound having thestructure (1):

or an enantiomer, optical isomer, diastereomer, N-oxide, crystallineform, hydrate, or pharmaceutically acceptable salt thereof.

A further embodiment of the present invention is a pharmaceuticalcomposition comprising a pharmaceutically acceptable carrier and atherapeutically effective amount of a compound according to the presentinvention.

Yet another embodiment of the present invention is a method formodulating blood glucose levels in a subject comprising administering toa subject an effective amount of a compound according to the presentinvention.

An additional embodiment of the present invention is a method forpreventing, treating, or ameliorating the effects of diabetes in asubject comprising administering to a subject an effective amount of acompound according to the present invention.

A further embodiment of the present invention is a method forpreventing, treating, or ameliorating the effects of hyperglycemiacomprising administering to a subject an effective amount of a compoundaccording to the present invention.

Yet another embodiment of the present invention is a method formodulating blood glucose levels in a subject comprising administering toa subject an effective amount of a pharmaceutical composition accordingto the present invention.

Another embodiment of the present invention is a method for preventing,treating, or ameliorating the effects of diabetes in a subjectcomprising administering to a subject an effective amount of apharmaceutical composition according to the present invention.

A further embodiment of the present invention is a method forpreventing, treating, ameliorating the effects of hyperglycemiacomprising administering to a subject an effective amount of apharmaceutical composition according to the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 demonstrates that tetrabenazine reduces the blood glucoseexcursion during an intraperitoneal glucose tolerance test (IPGTT). FIG.1A shows the blood glucose values during IPGTT of Lewis rats treatedwith vehicle alone (open symbol) or with tetrabenazine at the indicateddoses. Error bars indicate the standard error of the mean (S.E.M.)(n=25) at the indicated dose (closed symbols). FIG. 1B shows the resultspresented as AUC (area under the curve) IPGTT. The asterisk indicatesAUC IPGTT for vehicle controls (n=25) was significantly higher than theAUC IPGTT of the same TBZ treated animals (n=25) (p<0.05). The doubleasterisk indicates that the AUC IPGTT following treatment with TBZ andL-DOPA was significantly different than that of TBZ alone (n=6). Errorbars represent S.E.M.

FIG. 2 demonstrates that TBZ reduces the dopamine content of brain (A)and pancreas (B) tissue. Tetrabenazine at 1.5 mg/Kg body weight wasadministered intravenously (i.v.) to Lewis rats. One hour later, theanimals were euthanized and the brains and pancreata harvested andextracted in buffer. The dopamine concentration in the extract wasdetermined by ELISA and normalized to the total protein content. Theerror bars represent the S.E.M. from measurements of three TBZ treatedand three control Lewis rats. An asterisk represents a significantdifference (p<0.05) from control.

FIG. 3 demonstrates that tetrabenazine alters glucose stimulated insulinand glucagon secretion in vivo. Plasma insulin (FIGS. 3A and 3C) andglucagon concentrations (FIGS. 3B and 3D) were measured during IPGTT ofLewis rats (>11 weeks old) (n=6) treated with vehicle alone (opencolumns and circles). One week later, a second IPGTT was performed withTBZ (2.25 mg/Kg body weight) (filled columns and circles), TBZ (2.25mg/Kg body weight) plus dopamine (6.0 mg/Kg), or following five dailyinjections TBZ (0.3 mg/Kg body weight). An asterisk represents asignificant difference (p<0.05) from control.

FIG. 4 demonstrates that DTBZ enhances glucose stimulated insulinsecretion in rat islets ex vivo. Hand picked purified islets werecultured in high or low glucose containing media with and without DTBZ.Serial insulin concentration measurements in the supernatant wereperformed and the means and S.E.M. calculated. An asterisk represents asignificant difference (p<0.05) from control.

FIG. 5 shows VMAT2 mRNA and protein in islets and pancreas of controland streptozotocin treated Lewis rats. FIG. 5A shows the products of theqRT-PCR assay on total RNA from: brain, purified islets and totalpancreas using VMAT2 primers that specifically amplify a 175 base pair(bp) fragment. Untranscribed RNA from purified islets was used as acontrol. GelPilot 200 bp ladder was the molecular weight standard. FIG.5B shows the relative accumulation of VMAT2 mRNA in pancreata of controland streptozotocin-induced diabetic Lewis rats. The average accumulationof VMAT2 mRNA in streptozotocin-treated rodents was approximatelyeight-fold lower than the average accumulation in untreated pancreata(p<0.005) FIG. 5C shows a Western blot analysis of VMAT2 expression inprotein lysates prepared from control and streptozotocin-induceddiabetic Lewis rats.

FIG. 6 shows the structures of TBZ, DTBZ and compound 1.

FIG. 7 shows the results of the glucose tolerance tests of novelhypoglycemic compounds. Six hours fasted Lewis rats were administeredvarious compounds intravenously (30 minutes, 2 mg/kg) followed byintraperitoneal glucose injection (0 minute, 2 g/kg), and blood glucoselevels were monitored for 120 minutes. FIG. 7A shows the blood glucoseconcentration versus time curves for three repeat experiments withvehicle, TBZ, and compound 8 performed in a single rodent. FIG. 7B showsAUC IPGTT for a number of compounds according to the present invention.The area under the curve (AUC) IPGTT was calculated by the trapezoidalrule. Error bars indicate S.E.M. (n=5). The statistical significance ofthe difference between drug and vehicle was calculated by the method ofStudent. A p value of less than 0.005 is shown by an asterisk. The pvalue of the difference between the average AUC IPGTT of TBZ andcompound 8 was 0.030.

FIG. 8 shows the effect, post-injection, of compound 8 onbiodistribution of [¹¹C]DTBZ in pancreas. Positron emission tomography(PET) scans were performed on anesthetized 12-14-week-old Lewis malerats injected with radioligand [¹¹C]DTBZ (0 minute, 0.5-1.0 μCi/gm bodyweight, specific activity >2000 mCi/mol) and cold compound 8 (30minutes, 2 mg/kg). The fraction of radioligand in pancreas relative tothe total amount of injected was calculated and plotted versus timeafter injection. Error bars indicate S.E.M. (n=3). The area under thecurve (AUC) from 30 minutes to 90 minutes was calculated by thetrapezoidal rule for each experiment. The statistical significance ofthe difference between the average AUC baseline and AUC compound 8 wasp>0.05 as calculated by the method of Student.

FIG. 9 is a graph showing the quantitation of [¹¹C]DTBZ uptake in Lewisrat pancreas.

FIG. 10 is a graph showing % inhibition of control fluorescence in ahigh throughput screen according to the present invention.

FIG. 11 shows the synthetic scheme of compound 1. The first number undera structure sets forth the compound number. For example, “1” indicatescompound 1.

FIG. 12 shows the synthetic schemes of analogs of compound 8. The firstnumber under a structure sets forth the compound number.

DETAILED DESCRIPTION OF THE INVENTION

One embodiment of the present invention is a compound of formula I:

-   -   wherein R₁, R₂, R₃, R₅, and R₇ are independently selected from        H, halogen, hydroxyl, C₁₋₈alkyl, C₁₋₈alkenyl, C₁₋₈alkynyl,        C₁₋₈alkoxy, 3- to 8-membered carbocyclic or heterocyclic, aryl,        heteroaryl, C₁₋₄aralkyl, residues of glycolic acid, ethylene        glycol/propylene glycol copolymers, carboxylate, ester, amide,        carbohydrate, amino acid, alditol, OC(X)₂COOH, SC(X)₂COOH,        NHCHXCOOH, COY, CO₂Y, sulfate, sulfonamide, sulfoxide,        sulfonate, sulfone, thioalkyl, thioester, propylphthalimide, and        thioether;    -   R₄ and R₅, which are attached to one or more positions of at        least one carbon atom of the respective rings, are independently        selected from H, halogen, hydroxyl, C₁₋₈alkyl, C₁₋₈alkenyl,        C₁₋₈alkynyl, C₁₋₈alkoxy, 3- to 8-membered carbocyclic or        heterocyclic, aryl, heteroaryl, C₁-4aralkyl, residues of        glycolic acid, ethylene glycol/propylene glycol copolymers,        carboxylate, ester, amide, carbohydrate, amino acid, alditol,        OC(X)₂COOH, SC(X)₂COOH, NHCHXCOOH, COY, CO₂Y, sulfate,        sulfonamide, sulfoxide, sulfonate, sulfone, thioalkyl,        thioester, propylphthalimide, and thioether;    -   X is selected from H, C₁₋₈alkyl, C₁₋₈alkenyl, C₁₋₈alkynyl,        carbocycle, aryl, heteroaryl, heterocycle, alkylaryl,        alkylheteroaryl, and alkylheterocycle, wherein each alkyl,        carbocycle, aryl, heteroaryl, heterocycle, alkylaryl,        alkylheteroaryl, and alkylheterocycle may be optionally        substituted with at least one substituent;    -   Y is selected from H, C₁₋₈alkyl, C₁₋₈alkenyl, C₁₋₈alkynyl, aryl,        carbocycle, heteroaryl, heterocycle, alkylaryl, alkylheteroaryl,        alkylheterocycle, and heteroaromatic, wherein each alkyl,        alkenyl, alkynyl, aryl, carbocycle, heteroaryl, heterocycle,        alkylaryl, alkylheteroaryl, alkylheterocycle, and heteroaromatic        may be optionally substituted with at least one substituent; and    -   --- is an optional bond, wherein the optional bond is a single        bond or a double bond;        or an enantiomer, optical isomer, diastereomer, N-oxide,        crystalline form, hydrate, or pharmaceutically acceptable salt        thereof. In the present invention, it is to be understood that        each R group (e.g., R₁-R₉) includes all possible combinations of        the specific members recited in each R group.

In one aspect of this embodiment, the compound has formula II:

-   -   wherein R₁, R₂, R₃, R₈ and R₉ are independently selected from        the group consisting of H, halogen, C₁-C₈ alkyl, C₁-C₈alkenyl,        and C₁-C₈alkynyl;    -   R₄ and R₅, which are attached to one or more positions of at        least one carbon atom of the respective rings, are independently        selected from H, halogen, hydroxyl, C₁₋₈alkyl, C₁₋₈alkenyl,        C₁₋₈alkynyl, C₁₋₈alkoxy; and    -   --- is an optional bond, wherein the optional bond is a single        bond or a double bond;        or an enantiomer, optical isomer, diastereomer, N-oxide,        crystalline form, hydrate, or pharmaceutically acceptable salt        thereof.

Preferably, the compound has formula III:

-   -   wherein R₁, R₂, and R₃ are independently selected from the group        consisting of H, C₁-C₈alkyl, C₁-C₈alkenyl, and C₁-C₈alkynyl; and    -   --- is an optional bond, wherein the optional bond is a single        bond or a double bond;        or an enantiomer, optical isomer, diastereomer, N-oxide,        crystalline form, hydrate, or pharmaceutically acceptable salt        thereof.

More preferably, the compound has formula III, wherein R₁ is selectedfrom the group consisting of H, C₁-C₈alkyl, C₁-C₈alkenyl, andC₁-C₈alkynyl; R₂ and R₃ are selected from the group consisting of H andC₁alkyl; and --- is a single bond if R₂ or R₃ is not H.

In another preferred embodiment, the compound is compound 15:

or an enantiomer, optical isomer, diastereomer, N-oxide, crystallineform, hydrate, or pharmaceutically acceptable salt thereof.

In another aspect of this embodiment, the compound has formula IV:

-   -   wherein R₁, R₈, and R₉ are independently selected from the group        consisting of H, halogen, hydroxyl, C₁₋₈alkyl, C₁₋₈alkenyl,        C₁₋₈alkynyl, C₁₋₈alkoxy, 3- to 8-membered carbocyclic or        heterocyclic, aryl, heteroaryl, C₁₋₄aralkyl, residues of        glycolic acid, ethylene glycol/propylene glycol copolymers,        carboxylate, ester, amide, carbohydrate, amino acid, alditol,        OC(X)₂COOH, SC(X)₂COOH, NHCHXCOOH, COY, CO₂Y, sulfate,        sulfonamide, sulfoxide, sulfonate, sulfone, thioalkyl,        thioester, propylphthalimide, and thioether;    -   X is selected from H, C₁₋₈alkyl, C₁₋₈alkenyl, C₁₋₈alkynyl,        carbocycle, aryl, heteroaryl, heterocycle, alkylaryl,        alkylheteroaryl, and alkylheterocycle, wherein each alkyl,        carbocycle, aryl, heteroaryl, heterocycle, alkylaryl,        alkylheteroaryl, and alkylheterocycle may be optionally        substituted with at least one substituent;    -   Y is selected from H, C₁₋₈alkyl, C₁₋₈alkenyl, C₁₋₈alkynyl, aryl,        carbocycle, heteroaryl, heterocycle, alkylaryl, alkylheteroaryl,        alkylheterocycle, and heteroaromatic, wherein each alkyl,        alkenyl, alkynyl, aryl, carbocycle, heteroaryl, heterocycle,        alkylaryl, alkylheteroaryl, alkylheterocycle, and heteroaromatic        may be optionally substituted with at least one substituent;        or an enantiomer, optical isomer, diastereomer, N-oxide,        crystalline form, hydrate, or pharmaceutically acceptable salt        thereof.

Non-limiting examples of a compound according to the present inventioninclude:

-   -   (1) a compound having formula IV, wherein R₁ is —CH₂—CH—(CH₃)₂        and R₈, and R₉ are as set forth in the previous paragraph;    -   (2) a compound having formula IV, wherein R₈ is methyl, and R₁        and R₉ are as set forth in the previous paragraph;    -   (3) a compound having formula IV, wherein R₉ is methyl, and        wherein R₁ and R₈ are as set forth in the previous paragraph;    -   (4) a compound having formula IV, wherein both R₈ and R₉ are        methyl, and R₁ is as set forth in the previous paragraph;    -   (5) a compound having formula IV, wherein R₁ is selected from        C₁₋₈alkyl, C₁₋₈alkenyl, C₁₋₈alkynyl, C₁₋₈alkoxy, and R₈ and R₉        are both methyl;    -   (6) compound 8:

-   -   (7) an enantiomer, optical isomer, diastereomer, N-oxide,        crystalline form, hydrate, or pharmaceutically acceptable salt        of any of the compounds listed in (1)-(6).

Another embodiment of the present invention is a compound having thestructure (1):

or an enantiomer, optical isomer, diastereomer, N-oxide, crystallineform, hydrate, or pharmaceutically acceptable salt thereof.

A further embodiment of the present invention is a pharmaceuticalcomposition comprising a pharmaceutically acceptable carrier and atherapeutically effective amount of a compound according to the presentinvention, including those compounds set forth above. Preferably, thepharmaceutical composition includes a therapeutically acceptable amountof a compound according to formula I, compound 8, compound 15, orcombinations thereof.

Yet another embodiment of the present invention is a method formodulating blood glucose levels in a subject. This method comprisesadministering to a subject an effective amount of a compound or apharmaceutical composition according to the present invention.Preferably, the compound is a compound of formula I, compound 8,compound 15, or combinations thereof. Preferably, the pharmaceuticalcomposition includes a therapeutically acceptable amount of a compoundaccording to formula I, compound 8, compound 15, or combinationsthereof. In one aspect of this embodiment, the compound orpharmaceutical composition acts by interacting with, e.g., binding to,VMAT2 to provide the modulation.

As used herein in relation to blood glucose levels, “modulate,”“modulating,” and like terms mean to increase or, preferably, todecrease the blood glucose levels in a mammal, e.g., a human patient,administered a compound or pharmaceutical composition according to thepresent invention relative to a patient who is not administered thecompound.

An additional embodiment of the present invention is a method forpreventing, treating, or ameliorating the effects of diabetes (such astype I or type II diabetes) in a subject. This method comprisesadministering to a subject an effective amount of a compound or apharmaceutical composition according to the present invention.Preferably, the compound is a compound of formula I, compound 8,compound 15, or combinations thereof. Preferably, the pharmaceuticalcomposition includes a therapeutically acceptable amount of a compoundaccording to formula I, compound 8, compound 15, or combinationsthereof. In one aspect of this embodiment, the compound orpharmaceutical composition acts by interacting with, e.g., binding to,VMAT2 to prevent, treat, or ameliorate the effects of diabetes.

A further embodiment of the present invention is a method forpreventing, treating, or ameliorating the effects of hyperglycemia. Thismethod comprises administering to a subject an effective amount of acompound or a pharmaceutical composition according to the presentinvention. Preferably, the compound is a compound of formula I, compound8, compound 15, or combinations thereof. Preferably, the pharmaceuticalcomposition includes a therapeutically acceptable amount of a compoundaccording to formula I, compound 8, compound 15, or combinationsthereof. In one aspect of this embodiment, the compound orpharmaceutical composition acts by interacting with, e.g., binding to,VMAT2 to prevent, treat, or ameliorate the effects of hyperglycemia.

In the present invention, an “effective” amount or “therapeuticallyeffective” amount of a compound or a pharmaceutical composition is anamount of such a compound or a pharmaceutical composition that issufficient to effect beneficial or desired results as described hereinwhen administered to a subject such as a mammal, preferably a human, inneed of such therapy, e.g., who is suffering from diabetes orhyperglycemia. Effective dosage forms, modes of administration, anddosage amounts may be determined empirically, and making suchdeterminations is within the skill of the art. It is understood by thoseskilled in the art that the dosage amount will vary with the route ofadministration, the rate of excretion, the duration of the treatment,the identity of any other drugs being administered, the age, size, andspecies of mammal, e.g., human patient, and like factors well known inthe arts of medicine and veterinary medicine. In general, a suitabledose of a compound or a pharmaceutical composition according to theinvention will be that amount of the compound or the pharmaceuticalcomposition, which is the lowest dose effective to produce the desiredeffect with no or minimal side effects.

Suitable, non-limiting examples of dosages of a compound orpharmaceutical composition according to the present invention is fromabout 1 ng/kg to about 1000 mg/kg, such as from about 0.1-1.0 μg/gm,including 0.20-0.80 μg/gm, as well as e.g., about 1 mg/kg to about 100mg/kg, including from about 5 mg/kg to about 50 mg/kg. Otherrepresentative dosages of a compound or a pharmaceutical compositionaccording to the present invention include about 1 mg/kg, 5 mg/kg, 10mg/kg, 15 mg/kg, 20 mg/kg, 25 mg/kg, 30 mg/kg, 35 mg/kg, 40 mg/kg, 45mg/kg, 50 mg/kg, 60 mg/kg, 70 mg/kg, 80 mg/kg, 90 mg/kg, 100 mg/kg, 125mg/kg, 150 mg/kg, 175 mg/kg, 200 mg/kg, 250 mg/kg, 300 mg/kg, 400 mg/kg,500 mg/kg, 600 mg/kg, 700 mg/kg, 800 mg/kg, 900 mg/kg, 1000 mg/kg orthose dosages disclosed in the present Examples. The effective dose of acompound/pharmaceutical composition may be administered as two, three,four, five, six or more sub-doses, administered separately atappropriate intervals throughout the day.

A pharmaceutical composition of the present invention may beadministered in any desired and effective manner: for oral ingestion, oras an ointment or drop for local administration to the eyes, or forparenteral or other administration in any appropriate manner such asintraperitoneal, subcutaneous, topical, intradermal, inhalation,intrapulmonary, rectal, vaginal, sublingual, intramuscular, intravenous,intraarterial, intrathecal, or intralymphatic. Further, a pharmaceuticalcomposition of the present invention may be administered in conjunctionwith other treatments. A pharmaceutical composition of the presentinvention may be encapsulated or otherwise protected against gastric orother secretions, if desired.

The pharmaceutically acceptable compositions of the invention compriseone or more active ingredients in admixture with one or morepharmaceutically-acceptable carriers and, optionally, one or more othercompounds, drugs, ingredients and/or materials. Regardless of the routeof administration selected, the agents/compounds of the presentinvention are formulated into pharmaceutically-acceptable dosage formsby conventional methods known to those of skill in the art. See, e.g.,Remington, The Science and Practice of Pharmacy (21^(st) Edition,Lippincott Williams and Wilkins, Philadelphia, Pa.).

Pharmaceutically acceptable carriers are well known in the art (see,e.g., Remington, The Science and Practice of Pharmacy (21^(st) Edition,Lippincott Williams and Wilkins, Philadelphia, Pa.) and The NationalFormulary (American Pharmaceutical Association, Washington, D.C.)) andinclude sugars (e.g., lactose, sucrose, mannitol, and sorbitol),starches, cellulose preparations, calcium phosphates (e.g., dicalciumphosphate, tricalcium phosphate and calcium hydrogen phosphate), sodiumcitrate, water, aqueous solutions (e.g., saline, sodium chlorideinjection, Ringer's injection, dextrose injection, dextrose and sodiumchloride injection, lactated Ringer's injection), alcohols (e.g., ethylalcohol, propyl alcohol, and benzyl alcohol), polyols (e.g., glycerol,propylene glycol, and polyethylene glycol), organic esters (e.g., ethyloleate and tryglycerides), biodegradable polymers (e.g.,polylactide-polyglycolide, poly(orthoesters), and poly(anhydrides)),elastomeric matrices, liposomes, microspheres, oils (e.g., corn, germ,olive, castor, sesame, cottonseed, and groundnut), cocoa butter, waxes(e.g., suppository waxes), paraffins, silicones, talc, silicylate, etc.Each pharmaceutically acceptable carrier used in a pharmaceuticalcomposition of the invention must be “acceptable” in the sense of beingcompatible with the other ingredients of the formulation and notinjurious to the subject. Carriers suitable for a selected dosage formand intended route of administration are well known in the art, andacceptable carriers for a chosen dosage form and method ofadministration can be determined using ordinary skill in the art.

The pharmaceutical compositions of the invention may, optionally,contain additional ingredients and/or materials commonly used in suchpharmaceutical compositions. These ingredients and materials are wellknown in the art and include (1) fillers or extenders, such as starches,lactose, sucrose, glucose, mannitol, and silicic acid; (2) binders, suchas carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone,hydroxypropylmethyl cellulose, sucrose and acacia; (3) humectants, suchas glycerol; (4) disintegrating agents, such as agar-agar, calciumcarbonate, potato or tapioca starch, alginic acid, certain silicates,sodium starch glycolate, cross-linked sodium carboxymethyl cellulose andsodium carbonate; (5) solution retarding agents, such as paraffin; (6)absorption accelerators, such as quaternary ammonium compounds; (7)wetting agents, such as cetyl alcohol and glycerol monostearate; (8)absorbents, such as kaolin and bentonite clay; (9) lubricants, such astalc, calcium stearate, magnesium stearate, solid polyethylene glycols,and sodium lauryl sulfate; (10) suspending agents, such as ethoxylatedisostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters,microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agarand tragacanth; (11) buffering agents; (12) excipients, such as lactose,milk sugars, polyethylene glycols, animal and vegetable fats, oils,waxes, paraffins, cocoa butter, starches, tragacanth, cellulosederivatives, polyethylene glycol, silicones, bentonites, silicic acid,talc, salicylate, zinc oxide, aluminum hydroxide, calcium silicates, andpolyamide powder; (13) inert diluents, such as water or other solvents;(14) preservatives; (15) surface-active agents; (16) dispersing agents;(17) control-release or absorption-delaying agents, such ashydroxypropylmethyl cellulose, other polymer matrices, biodegradablepolymers, liposomes, microspheres, aluminum monosterate, gelatin, andwaxes; (18) opacifying agents; (19) adjuvants; (20) wetting agents; (21)emulsifying and suspending agents; (22), solubilizing agents andemulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate,ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol,1,3-butylene glycol, oils (in particular, cottonseed, groundnut, corn,germ, olive, castor and sesame oils), glycerol, tetrahydrofuryl alcohol,polyethylene glycols and fatty acid esters of sorbitan; (23)propellants, such as chlorofluorohydrocarbons and volatile unsubstitutedhydrocarbons, such as butane and propane; (24) antioxidants; (25) agentswhich render the formulation isotonic with the blood of the intendedrecipient, such as sugars and sodium chloride; (26) thickening agents;(27) coating materials, such as lecithin; and (28) sweetening,flavoring, coloring, perfuming and preservative agents. Each suchingredient or material must be “acceptable” in the sense of beingcompatible with the other ingredients of the formulation and notinjurious to the subject. Ingredients and materials suitable for aselected dosage form and intended route of administration are well knownin the art, and acceptable ingredients and materials for a chosen dosageform and method of administration may be determined using ordinary skillin the art.

Pharmaceutical compositions suitable for oral administration may be inthe form of capsules, cachets, pills, tablets, powders, granules, asolution or a suspension in an aqueous or non-aqueous liquid, anoil-in-water or water-in-oil liquid emulsion, an elixir or syrup, apastille, a bolus, an electuary or a paste. These formulations may beprepared by methods known in the art, e.g., by means of conventionalpan-coating, mixing, granulation or lyophilization processes.

Solid dosage forms for oral administration (capsules, tablets, pills,dragees, powders, granules and the like) may be prepared, e.g., bymixing the active ingredient(s) with one or morepharmaceutically-acceptable carriers and, optionally, one or morefillers, extenders, binders, humectants, disintegrating agents, solutionretarding agents, absorption accelerators, wetting agents, absorbents,lubricants, and/or coloring agents. Solid compositions of a similar typemay be employed as fillers in soft and hard-filled gelatin capsulesusing a suitable excipient. A tablet may be made by compression ormolding, optionally with one or more accessory ingredients. Compressedtablets may be prepared using a suitable binder, lubricant, inertdiluent, preservative, disintegrant, surface-active or dispersing agent.Molded tablets may be made by molding in a suitable machine. Thetablets, and other solid dosage forms, such as dragees, capsules, pillsand granules, may optionally be scored or prepared with coatings andshells, such as enteric coatings and other coatings well known in thepharmaceutical-formulating art. They may also be formulated so as toprovide slow or controlled release of the active ingredient therein.They may be sterilized by, for example, filtration through abacteria-retaining filter. These compositions may also optionallycontain opacifying agents and may be of a composition such that theyrelease the active ingredient only, or preferentially, in a certainportion of the gastrointestinal tract, optionally, in a delayed manner.The active ingredient can also be in microencapsulated form.

Liquid dosage forms for oral administration includepharmaceutically-acceptable emulsions, microemulsions, solutions,suspensions, syrups and elixirs. The liquid dosage forms may containsuitable inert diluents commonly used in the art. Besides inertdiluents, the oral compositions may also include adjuvants, such aswetting agents, emulsifying and suspending agents, sweetening,flavoring, coloring, perfuming and preservative agents. Suspensions maycontain suspending agents.

Pharmaceutical compositions for rectal or vaginal administration may bepresented as a suppository, which may be prepared by mixing one or moreactive ingredient(s) with one or more suitable nonirritating carrierswhich are solid at room temperature, but liquid at body temperature and,therefore, will melt in the rectum or vaginal cavity and release theactive compound. Pharmaceutical compositions which are suitable forvaginal administration also include pessaries, tampons, creams, gels,pastes, foams or spray formulations containing suchpharmaceutically-acceptable carriers as are known in the art to beappropriate.

Dosage forms for the topical or transdermal administration includepowders, sprays, ointments, pastes, creams, lotions, gels, solutions,patches, drops and inhalants. The active agent(s)/compound(s) may bemixed under sterile conditions with a suitablepharmaceutically-acceptable carrier. The ointments, pastes, creams andgels may contain excipients. Powders and sprays may contain excipientsand propellants.

Pharmaceutical compositions suitable for parenteral administrationscomprise one or more agent(s)/compound(s) in combination with one ormore pharmaceutically-acceptable sterile isotonic aqueous or non-aqueoussolutions, dispersions, suspensions or emulsions, or sterile powderswhich may be reconstituted into sterile injectable solutions ordispersions just prior to use, which may contain suitable antioxidants,buffers, solutes which render the formulation isotonic with the blood ofthe intended recipient, or suspending or thickening agents. Properfluidity can be maintained, for example, by the use of coatingmaterials, by the maintenance of the required particle size in the caseof dispersions, and by the use of surfactants. These compositions mayalso contain suitable adjuvants, such as wetting agents, emulsifyingagents and dispersing agents. It may also be desirable to includeisotonic agents. In addition, prolonged absorption of the injectablepharmaceutical form may be brought about by the inclusion of agentswhich delay absorption.

In some cases, in order to prolong the effect of a drug (e.g.,pharmaceutical formulation), it is desirable to slow its absorption fromsubcutaneous or intramuscular injection. This may be accomplished by theuse of a liquid suspension of crystalline or amorphous material havingpoor water solubility.

The rate of absorption of the active agent/drug then depends upon itsrate of dissolution which, in turn, may depend upon crystal size andcrystalline form. Alternatively, delayed absorption of aparenterally-administered agent/drug may be accomplished by dissolvingor suspending the active agent/drug in an oil vehicle. Injectable depotforms may be made by forming microencapsule matrices of the activeingredient in biodegradable polymers. Depending on the ratio of theactive ingredient to polymer, and the nature of the particular polymeremployed, the rate of active ingredient release can be controlled. Depotinjectable formulations are also prepared by entrapping the drug inliposomes or microemulsions which are compatible with body tissue. Theinjectable materials can be sterilized for example, by filtrationthrough a bacterial-retaining filter.

The formulations may be presented in unit-dose or multi-dose sealedcontainers, for example, ampules and vials, and may be stored in alyophilized condition requiring only the addition of the sterile liquidcarrier, for example water for injection, immediately prior to use.Extemporaneous injection solutions and suspensions may be prepared fromsterile powders, granules and tablets of the type described above.

In the present invention, the following definitions apply.

As used herein, the term “acyl” has its art-recognized meaning andrefers to a group represented by the general formula hydrocarbylC(O)—,preferably alkylC(O)—.

As used herein, the term “acylamino” has its art-recognized meaning andrefers to an amino group substituted with an acyl group and may berepresented, for example, by the formula hydrocarbylC(O)NH—.

As used herein, the term “alditol” means any of a class of acyclicalcohols containing multiple hydroxyl groups, which are derived from amonosaccharide containing a terminal carbonyl group and having achemical formula of the form C_(n)(H₂O)_(n) by reduction of the carbonylfunctional group. Alditol includes, for example, sorbitol.

The term “alkoxy” refers to an alkyl group, preferably a lower alkylgroup, having an oxygen attached thereto. Representative alkoxy groupsinclude methoxy, ethoxy, propoxy, isopropoxy, tert-butoxy and the like.

The term “alkoxyalkyl” refers to an alkyl group substituted with analkoxy group and may be represented by the general formulaalkyl-O-alkyl.

The term “alkenyl”, as used herein, refers to an aliphatic groupcontaining at least one double bond and is intended to include both“unsubstituted alkenyls” and “substituted alkenyls”, the latter of whichrefers to alkenyl moieties having substituents replacing a hydrogen onone or more carbons of the alkenyl group. Such substituents may occur onone or more carbons that are included or not included in one or moredouble bonds. Moreover, such substituents include all those contemplatedfor alkyl groups, as discussed below, except where stability isprohibitive. For example, substitution of alkenyl groups by one or morealkyl, carbocyclyl, aryl, heterocyclyl, or heteroaryl groups iscontemplated.

The term “alkyl” refers to the radical of saturated aliphatic groups,including straight-chain alkyl groups, branched-chain alkyl groups,cycloalkyl (alicyclic) groups, alkyl-substituted cycloalkyl groups, andcycloalkyl-substituted alkyl groups. In preferred embodiments, astraight chain or branched chain alkyl has 8 or fewer carbon atoms inits backbone (e.g., C₁-C₈ for straight chains, C₃-C₈ for branchedchains). Likewise, preferred cycloalkyls have from 3-8 carbon atoms intheir ring structure, including 5, 6 or 7 carbons in the ring structure.

Moreover, unless otherwise indicated, the term “alkyl” (or “loweralkyl”) as used throughout the specification, examples, and claims isintended to include both “unsubstituted alkyls” and “substitutedalkyls”, the latter of which refers to alkyl moieties havingsubstituents replacing a hydrogen on one or more carbons of thehydrocarbon backbone. Such substituents can include, for example, ahalogen, a hydroxyl, a carbonyl (such as a carboxyl, an alkoxycarbonyl,a formyl, or an acyl), a thiocarbonyl (such as a thioester, athioacetate, or a thioformate), an alkoxyl, a phosphoryl, a phosphate, aphosphonate, a phosphinate, an amino, an amido, an amidine, an imine, acyano, a nitro, an azido, a sulfhydryl, an alkylthio, a sulfate, asulfonate, a sulfamoyl, a sulfonamido, a sulfonyl, a heterocyclyl, anaralkyl, an aromatic, or heteroaromatic moiety. It will be understood bythose skilled in the art that the moieties substituted on thehydrocarbon chain can themselves be substituted, if appropriate. Forinstance, the substituents of a substituted alkyl may includesubstituted and unsubstituted forms of amino, azido, imino, amido,phosphoryl (including phosphonate and phosphinate), sulfonyl (includingsulfate, sulfonamido, sulfamoyl and sulfonate), and silyl groups, aswell as ethers, alkylthios, carbonyls (including ketones, aldehydes,carboxylates, and esters), —CF₃, —CN and the like. Exemplary substitutedalkyls are described below. Cycloalkyls can be further substituted withalkyls, alkenyls, alkoxys, alkylthios, aminoalkyls, carbonyl-substitutedalkyls, —CF₃, —CN, and the like.

The term “C_(x-y)” when used in conjunction with a chemical moiety, suchas, alkyl, alkenyl, or alkoxy is meant to include groups that containfrom x to y carbons in the chain. For example, the term “C_(x-y)alkyl”refers to substituted or unsubstituted saturated hydrocarbon groups,including straight-chain alkyl and branched-chain alkyl groups thatcontain from x to y carbons in the chain, including haloalkyl groupssuch as trifluoromethyl and 2,2,2-trifluoroethyl, etc. The terms“C_(2-y)alkenyl” and “C_(2-y)alkynyl” refer to substituted orunsubstituted unsaturated aliphatic groups analogous in length andpossible substitution to the alkyls described above, but that contain atleast one double or triple bond respectively.

The term “alkylaryl”, as used herein, refers to an aryl groupsubstituted with an alkyl group.

The term “alkylheteroaryl”, as used herein, refers to a heteroaryl groupsubstituted with an alkyl group.

The term “alkylheterocycle”, as used herein, refers to an heterocyclegroup substituted with an alkyl group.

The term “alkylthio”, as used herein, refers to a thiol groupsubstituted with an alkyl group and may be represented by the generalformula alkylS—.

The term “alkynyl”, as used herein, refers to an aliphatic groupcontaining at least one triple bond and is intended to include both“unsubstituted alkynyls” and “substituted alkynyls”, the latter of whichrefers to alkynyl moieties having substituents replacing a hydrogen onone or more carbons of the alkynyl group. Such substituents may occur onone or more carbons that are included or not included in one or moretriple bonds. Moreover, such substituents include all those contemplatedfor alkyl groups, as discussed above, except where stability isprohibitive. For example, substitution of alkynyl groups by one or morealkyl, carbocyclyl, aryl, heterocyclyl, or heteroaryl groups iscontemplated.

The term “amide”, as used herein, refers to a group

wherein R¹⁰ and R¹¹ each independently represent a hydrogen orhydrocarbyl group, or R¹⁰ and R¹¹ taken together with the N atom towhich they are attached complete a heterocycle having from 4 to 8 atomsin the ring structure.

The terms “amine” and “amino” are art-recognized and refer to bothunsubstituted and substituted amines and salts thereof, e.g., a moietythat can be represented by

wherein R¹⁰, R¹¹, and R11′ each independently represent a hydrogen or ahydrocarbyl group, or R¹⁰ and R¹¹ taken together with the N atom towhich they are attached complete a heterocycle having from 4 to 8 atomsin the ring structure.

The term “amino acid,” as used herein, refers a functional groupcontaining both amine and carboxyl groups.

The term “aminoalkyl”, as used herein, refers to an alkyl groupsubstituted with an amino group.

The term “aralkyl”, as used herein, refers to an alkyl group substitutedwith an aryl group.

The term “aryl” as used herein includes substituted or unsubstitutedsingle-ring aromatic groups in which each atom of the ring is carbon.Preferably the ring is a 3- to 8-membered ring, more preferably a6-membered ring. The term “aryl” also includes polycyclic ring systemshaving two or more cyclic rings in which two or more carbons are commonto two adjoining rings wherein at least one of the rings is aromatic,e.g., the other cyclic rings can be cycloalkyls, cycloalkenyls,cycloalkynyls, aryls, heteroaryls, and/or heterocyclyls. Aryl groupsinclude benzene, naphthalene, phenanthrene, phenol, aniline, and thelike.

The term “carbamate” is art-recognized and refers to a group

wherein R¹⁰ and R¹¹ independently represent hydrogen or a hydrocarbylgroup.

The terms “carbocycle”, “carbocyclyl”, and “carbocyclic”, as usedherein, refer to a non-aromatic saturated or unsaturated ring in whicheach atom of the ring is carbon. Preferably a carbocycle ring containsfrom 3 to 8 atoms, including 5 to 7 atoms, such as for example, 6 atoms.

The term “carbohydrate”, as used herein, is a functional group thatincludes an aldehyde or ketone group with many hydroxyl groups added tothe carbon backbone, usually one on each carbon atom that is not part ofthe aldehyde or ketone functional group. Carbohydrate includemonosaccharides such as glucose, galactose, and fructose. Carbohydratealso include oligosaccharides made of two or more monosaccharides, butpreferably two monosaccharides.

The terms “carboxy” and “carboxyl”, as used herein, refer to a grouprepresented by the formula —CO₂H.

The term “carboxylate” refers to the conjugate base of a carboxyl group,represented by the formula —COO⁻.

The term “ester”, as used herein, refers to a group —C(O)OR¹⁰ whereinR¹⁰ represents a hydrocarbyl group.

The term “ether”, as used herein, refers to a hydrocarbyl group linkedthrough an oxygen to another hydrocarbyl group. Accordingly, an ethersubstituent of a hydrocarbyl group may be hydrocarbyl-O—. Ethers may beeither symmetrical or unsymmetrical. Examples of ethers include, but arenot limited to, heterocycle-O-heterocycle and aryl-O-heterocycle. Ethersinclude “alkoxyalkyl” groups, which may be represented by the generalformula alkyl-O-alkyl.

The terms “halo” and “halogen” are used interchangeably herein and meanhalogen and include chloro, fluoro, bromo, and iodo.

The term “heteroaryl” includes substituted or unsubstituted aromaticsingle ring structures, preferably 3- to 8-membered rings, morepreferably 5- to 7-membered rings, even more preferably 5- to 6-memberedrings, whose ring structures include at least one heteroatom, preferablyone to four heteroatoms, more preferably one or two heteroatoms. Theterm “heteroaryl” also includes polycyclic ring systems having two ormore cyclic rings in which two or more carbons are common to twoadjoining rings wherein at least one of the rings is heteroaromatic,e.g., the other cyclic rings can be cycloalkyls, cycloalkenyls,cycloalkynyls, aryls, heteroaryls, and/or heterocyclyls. Heteroarylgroups include, for example, pyrrole, furan, thiophene, imidazole,oxazole, thiazole, pyrazole, pyridine, pyrazine, pyridazine, andpyrimidine, and the like.

The term “heteroatom” as used herein means an atom of any element otherthan carbon or hydrogen. Preferred heteroatoms are nitrogen, oxygen, andsulfur.

The term “heteroaromatic” means at least one carbon atoms in thearomatic group is substituted with a heteroatom.

The terms “heterocyclyl”, “heterocycle”, “heterocyclic”, and the likerefer to substituted or unsubstituted non-aromatic ring structures,preferably 3- to 8-membered rings, whose ring structures include atleast one heteroatom, preferably one to four heteroatoms, morepreferably one or two heteroatoms. The terms “heterocyclyl,”“heterocyclic,” and the like also include polycyclic ring systems havingtwo or more cyclic rings in which two or more carbons are common to twoadjoining rings wherein at least one of the rings is heterocyclic, e.g.,the other cyclic rings can be cycloalkyls, cycloalkenyls, cycloalkynyls,aryls, heteroaryls, and/or heterocyclyls. Heterocyclyl groups include,for example, piperidine, piperazine, pyrrolidine, morpholine, lactones,lactams, and the like.

The term “hydrocarbyl”, as used herein, refers to a group that is bondedthrough a carbon atom that does not have a ═O or ═S substituent, andtypically has at least one carbon-hydrogen bond and a primarily carbonbackbone, but may optionally include heteroatoms. Thus, groups likemethyl, ethoxyethyl, 2-pyridyl, and trifluoromethyl are considered to behydrocarbyl for the purposes of this application, but substituents suchas acetyl (which has a ═O substituent on the linking carbon) and ethoxy(which is linked through oxygen, not carbon) are not. Hydrocarbyl groupsinclude, but are not limited to aryl, heteroaryl, carbocycle,heterocycle, alkyl, alkenyl, alkynyl, and combinations thereof.

The term “hydroxyalkyl”, as used herein, refers to an alkyl groupsubstituted with a hydroxy group.

The term “lower” when used in conjunction with a chemical moiety, suchas, acyl, alkyl, alkenyl, alkynyl, or alkoxy is meant to include groupswhere there are ten or fewer non-hydrogen atoms in the substituent,preferably eight or fewer, such as for example, from about 2 to 8 carbonatoms, including less than 6 carbon atoms. A “lower alkyl”, for example,refers to an alkyl group that contains ten or fewer carbon atoms,preferably eight or fewer. In certain embodiments, acyl, alkyl, alkenyl,alkynyl, or alkoxy substituents defined herein are respectively loweracyl, lower alkyl, lower alkenyl, lower alkynyl, or lower alkoxy,whether they appear alone or in combination with other substituents,such as in the recitations hydroxyalkyl and aralkyl (in which case, forexample, the atoms within the aryl group are not counted when countingthe carbon atoms in the alkyl substituent).

The terms “polycyclyl”, “polycycle”, and “polycyclic” refer to two ormore rings (e.g., cycloalkyls, cycloalkenyls, cycloalkynyls, aryls,heteroaryls, and/or heterocyclyls) in which two or more atoms are commonto two adjoining rings, e.g., the rings are “fused rings”. Each of therings of the polycycle can be substituted or unsubstituted. In certainembodiments, each ring of the polycycle contains from 3 to 10 atoms inthe ring, preferably from 3 to 8, such as for example, 5 to 7.

The term “substituted” refers to moieties having substituents replacinga hydrogen on one or more carbons of the backbone. It will be understoodthat “substitution” or “substituted with” includes the implicit provisothat such substitution is in accordance with the permitted valence ofthe substituted atom and the substituent, and that the substitutionresults in a stable compound, e.g., which does not spontaneously undergotransformation such as by rearrangement, cyclization, elimination, etc.As used herein, the term “substituted” is contemplated to include allpermissible substituents of organic compounds. In a broad aspect, thepermissible substituents include acyclic and cyclic, branched andunbranched, carbocyclic and heterocyclic, aromatic and non-aromaticsubstituents of organic compounds. The permissible substituents can beone or more and the same or different for appropriate organic compounds.For purposes of this invention, the heteroatoms such as nitrogen mayhave hydrogen substituents and/or any permissible substituents oforganic compounds described herein which satisfy the valences of theheteroatoms. Substituents can include any substituents described herein,for example, a halogen, a hydroxyl, a carbonyl (such as a carboxyl, analkoxycarbonyl, a formyl, or an acyl), a thiocarbonyl (such as athioester, a thioacetate, or a thioformate), an alkoxyl, a phosphoryl, aphosphate, a phosphonate, a phosphinate, an amino, an amido, an amidine,an imine, a cyano, a nitro, an azido, a sulfhydryl, an alkylthio, asulfate, a sulfonate, a sulfamoyl, a sulfonamido, a sulfonyl, aheterocyclyl, an aralkyl, or an aromatic or heteroaromatic moiety. Itwill be understood by those skilled in the art that the moietiessubstituted on the hydrocarbon chain can themselves be substituted, ifappropriate.

As used herein, the term “substituent,” means H, cyano, oxo, nitro,acyl, acylamino, halogen, hydroxy, amino acid, amine, amide, carbamate,ester, ether, carboxylic acid, thio, thioalkyl, thioester, thioether,C₁₋₈ alkyl, C₁₋₈alkoxy, C₁₋₈alkenyl, C₁₋₈aralkyl, 3- to 8-memberedcarbocyclic, 3- to 8-membered heterocyclic, 3- to 8-membered aryl, or 3-to 8-membered heteroaryl, sulfate, sulfonamide, sulfoxide, sulfonate,sulfone, alkylsulfonyl, and arylsulfonyl.

Unless specifically stated as “unsubstituted,” references to chemicalmoieties herein are understood to include substituted variants. Forexample, reference to an “aryl” group or moiety implicitly includes bothsubstituted and unsubstituted variants.

The term “sulfate” is art-recognized and refers to the group —OSO₃H, ora pharmaceutically acceptable salt thereof.

The term “sulfonamide” is art-recognized and refers to the grouprepresented by the general formulae

wherein R¹⁰ and R¹¹ independently represents hydrogen or hydrocarbyl.

The term “sulfoxide” is art-recognized and refers to the group —S(O)—R⁷,wherein R⁷ represents a hydrocarbyl.

The term “sulfonate” is art-recognized and refers to the group SO₃H, ora pharmaceutically acceptable salt thereof.

The term “sulfone” is art-recognized and refers to the group —S(O)₂—R⁷,wherein R⁷ represents a hydrocarbyl.

The term “thioalkyl”, as used herein, refers to an alkyl groupsubstituted with a thiol group.

The term “thioester”, as used herein, refers to a group —C(O)SR⁷ or—SC(O)R⁷ wherein R⁷ represents a hydrocarbyl.

The term “thioether”, as used herein, is equivalent to an ether, whereinthe oxygen is replaced with a sulfur.

The following examples are provided to further illustrate the methods ofthe present invention. These examples are illustrative only and are notintended to limit the scope of the invention in any way.

EXAMPLES Example 1 Materials and Methods Drugs and Reagents

L-epinephrine bitartrate, D-glucose, L-DOPA, sodium citrate wereobtained from Sigma Chemicals.

All cell culture media and supplements were obtained from Invitrogen(Carlsbad, Calif.). Tissue culture plates were obtained from Falconware(Becton-Dickinson, Inc., Oxnard, Calif.). Tetrabenazine anddihydrotetrabenazine were obtained from the National Institute of MentalHealth's Chemical Synthesis and Drug Supply Program or Tocris Bioscience(Ellisville, Mo.). All other chemicals (other than the compoundssynthesized as described below) were of the highest commercial qualityavailable.

Experimental Animals

All animal studies were reviewed and approved by the InstitutionalAnimal Care and Use Committee (IACUC) at Columbia University's Collegeof Physicians and Surgeons. All experiments were performed in accordancewith ‘Principles of laboratory animal care’ (NIH publication no. 85-23,revised 1985). Normal male Lewis rats were obtained from Taconic(Taconic Inc., Germantown, N.Y.) and were housed under conditions ofcontrolled humidity (55±5%), temperature (23±1° C.), and lighting (lighton from 6 a.m. to 6 p.m.) with free access to standard laboratory Purinarat chow and water.

Rats were handled daily to minimize nonspecific stress for more than 7days before the experiments began. In most experiments, it was necessaryto measure blood glucose in fasting animals. For these groups, food wasremoved at the beginning of the light cycle, 6 hours before glucoselevels were tested. Fasting rats for longer than eight hours resulted inhigher experimental variability.

Sixty minutes prior to intraperitoneal glucose tolerance testing(IPGTT), anesthesia of male Lewis rats was induced with isoflurane (3-4%in oxygen) and maintained with 1-2% isoflurane in oxygen.

Anaesthetized rats were administered TBZ at the indicated dose byintravenous (i.v.) injection using the penile vein. TBZ or L-DOPA wasdissolved in neat sterile DMSO and diluted (always more than 10 fold) insterile saline. Control rats received injections of vehicle alone (10%DMSO in Saline).

Animals were fully recovered for at least 30 minutes before receivingIPGTT. In specified experiments, L-DOPA was injected interperitoneal(I.P.) at the specified dose at the initiation of IPGTT. Abnormalglucose tolerance was induced by a single I.P. injection ofstreptozotocin (STZ) (Sigma Aldrich, St. Louis, Mo.) (50 mg/kg) toanimals that had been fasted 4 hours to enhance the efficacy of STZ.

STZ solution was prepared fresh by dissolving it in 0.1 M citrate buffer(pH 5.5) and terminally sterile filtered. Control Lewis age and weightmatched rats received a 0.5 ml/kg citrate vehicle alone viaintraperitoneal injection. The diabetic phenotype induced by STZ wasallowed to develop for one week before confirmation by glucose tolerancetesting. Animals were considered to have a stable diabetic phenotypeafter three consecutive measurements of blood glucose with a value ofgreater than 300 mg/dl. Animals failing this criterion were not used andeuthanized.

Blood Glucose, Insulin, Glucagon and Intraperitoneal Glucose ToleranceTests Measurements

Blood samples were collected between 12:00 noon and 2:00 p.m. from asuperficial blood vessel in the tails of the rats following 6 hours offasting. The fasting blood glucose (BG) levels of the rats were measuredusing an Accu-Check blood glucose monitoring system (Roche Diagnostics,Sommerville, N.J.). IPGTT was performed in 6-hour fasting anaesthetizedanimals as previously described (Weksler-Zangen, et al. 2001). Briefly,after baseline BG measurements, animals received an I.P. injection of 2grams of glucose/kg body weight. To minimize stress during theprocedure, rats were handled by the same operator during acclimatizationand later during weighing and IPGTT. Blood samples (50 μl or 150 μl)were collected at baseline and then again 15, 30, 60, 90, and 120minutes following I.P. injection of glucose. Blood glucoseconcentrations were measured immediately on these samples and theremainder processed. Plasma was immediately separated by centrifugationat 3,000×g for 15 minutes and then stored at −80° C. until analysis.

Insulin and glucagon concentration measurements in rat plasma wereperformed by ELISA as per the manufacturer's instructions using kitsfrom Linco Research Inc. (St. Charles, Mich.) and Alpco Diagnostics(Salem, N.H.), respectively. To validate the test, saline injectionswere performed by the same method. During this experiment, glucoseconcentration did not differ from baseline at each time point (data notshown). The area under the insulin, glucagon and IPGTT glucoseconcentration×time curve (AUC IPGTT) was calculated by the trapezoidalrule.

Islet Tissue and Glucose Stimulated Insulin Secretion

Rat pancreas digestion, islet isolations, and static insulin secretionassays were performed as previously described (Niswender et al. 2005;Sweet, et al. 2004; Sweet and Gilbert 2006). Purified islets werecultured in RPMI 1640 culture media with 10% fetal bovine serum at 37°C. in humidified air (5% CO₂) for 18 to 24 hours. Assessment of insulinsecretion in static media was carried out as follows. Islets werehandpicked twice into a Petri dishes containing Krebs-Ringer bicarbonate(KRB) buffer (with 3 mM glucose and 0.1% BSA) and pre-incubated for 60minutes (37° C. and 5% CO₂). Subsequently, batches of 100 islets (inquadruplicate) were transferred into 96-well plates containing 200 μl ofKRB with either 3 or 20 mM glucose, with or without 100 nM DTBZ andincubated for 60 minutes. The supernatant was removed and the insulinwas measured using ELISA (ALPCO, Windham, N.H.).

The Effect of DTBZ on Dipeptidyl Peptidase IV (DPP-IV)

The effect of DTBZ on DDP-IV was determined using the DPP profilingservice from BPS Bioscience, Inc. (San Diego, Calif.).

Dopamine Measurements

Anaesthetized rats received an intravenous injection of TBZ and weresacrificed one hour later.

Euthanasia was performed by exsanguination of the anesthetized animal.Brain and pancreas were harvested as quickly as possible and frozen at−80° C. until use. Frozen tissue was pulverized in a liquidnitrogen-cooled mortar and extracted in 0.01 N hydrochloric acid (HCl).The tissue extract was centrifuged at 10,000×g at 4° C. to remove debrisand the total protein was estimated by reading the absorbance at 280 nm.The concentration of dopamine in the extract was estimated using anELISA kit from Rocky Mountain Diagnostics (Colorado Springs, Colo.) asper the manufacturer's instructions and normalized to the extractprotein concentration.

Quantitation of VMAT2 mRNA in Pancreata and Islets of Lewis Rats.

Harvesting of pancreata was performed as follows. Anesthsized rats wereopened with a midline incision and the liver, stomach, and smallintestines reflected to expose the pancreas. The cavity was then bathedwith 10 ml of a 1:1 solution PBS 1× and RNAlater (Ambion, Austin, Tex.).The pancreas was dissected and transferred to a 50 ml polypropylene tubecontaining 6 ml of fresh RNAlater solution and—if not immediatelyprocessed—stored at −80° C. After thawing, the entire pancreas wastransferred into 1 ml of QIAzol (QIAGEN, Valencia, Calif.)/100 mg oftissue and homogenized. In the indicated experiments, purified and handpicked rat islets (about 500) were transferred directly to QIAzol. TotalRNA, either from pancreata or purified islets, was purified using theRNeasy Mini Kit (QIAGEN, Valencia, Calif.) in conjunction with theRNase-Free DNase Set (QIAGEN, Valencia, Calif.). All RNA extractionswere performed using RNase-/DNase-free laboratory ware. RNA wasquantified and assessed for purity by electrophoresis on a 1.6% agarosegel and UV spectrophotometry. Tissue processing, RNA extraction, andqRT-PCR assay set up were performed in separate designated laboratoryareas to prevent cross-contamination. All reverse transcriptasereactions were performed using the SuperScript III RT System fromINVITROGEN (Carlsbad, Calif.) with random-priming. The qPCR assays wereperformed using the amount of cDNA obtained by retro-transcribing 100 ngof total RNA.

The QuantiTect SYBR Green PCR Kit (INVITROGEN (Carlsbad, Calif.)) wasused to perform all the reactions in the presence of 0.2 μM primers, ina total volume of 25 μl. Samples were amplified with a precycling holdat 95° C. for 15 minutes, followed by 36 cycles of denaturation at 95°C. for 15 seconds, annealing at 55° C.-60° C. (depending from theprimers) for 30 seconds, and extension at 72° C. for 20 seconds. qRT-PCRreagent controls (reagents without any template or with 100 ng of notretro-transcribed RNA) were included in all the assays. Each assay wasrun in triplicate and repeated at least twice to verify the results, andthe mean copy number was used for analysis. The standard deviationbetween assays was not significant (5%) in all the experiments. Therelative amount of specific transcripts was calculated as previouslydescribed (Maffei, et al. 2004). To correct for sample to samplevariations in qRT-PCR efficiency and errors in sample quantitation, thelevel of both GAPDH transcripts and 18S rRNA was tested for use innormalization of specific RNA levels. In these experiments nosignificant differences were found between normalization by GAPDH mRNAlevel or normalization by 18S rRNA levels. All oligonucleotides weresynthesized by INVITROGEN (Carlsbad, Calif.). The primer sequences wereas follows: 5′-CGC AAA CTG ATC CTG TTC AT-3′ (VT2-2 F) (SEQ ID NO: 1)and 5′-AGA AGA TGC TTT CGG AGG TG-3′ (VT2-2 R) (SEQ ID NO: 2); 5′-AACGGA TTT GGC CGT ATC GGA C-3′ (rGAPDH F) (SEQ ID NO: 3) and 5′-TCG CTCCTG GAA GAT GGT GAT G-3′ (rGAPDH R) (SEQ ID NO: 4); 5′-TTS GAA CGT CTGCCC TAT CAA-3′ (rl8S F) (SEQ ID NO: 5) and 5′-CAA TTA CAG GGC CTC GAAAG-3′ (r18S R) (SEQ ID NO: 6). The relative amounts of mRNA, werecalculated by the comparative cycle threshold (CT) method described byLivak and Schmittgen (Livak and Schmittgen 2001).

Quantitation of VMAT2 and Preproinsulin Protein in Pancreas Lysates byWestern Blot.

Western blot analysis was conducted on pancreas tissue obtained fromcontrol and diabetic STZ treated rats using standard procedures.Briefly, sample tissues were flash frozen in liquid nitrogen and groundto a fine powder while frozen. Powdered proteins were solubilized in1×PBS; 1% Nonidet P-40; 0.5% sodium deoxycholate and 0.1% SDS. Acomplete cocktail of mammalian protease inhibitors (Sigma-Aldrich, St.Louis, Mo.), at high concentration, was added immediately prior tosample preparation. Protein concentrations were determined using aBio-Rad protein assay (Bio-Rad Inc., Hercules, Calif.) with bovine serumalbumin standards and following the manufacturers recommendations.Solubilized proteins were diluted in Laemmli sample buffer and incubatedat 100° C. for 1 minute. Protein separation was conducted using theBio-Rad Lab Mini-gel electrophoresis system on 15% Acrylamide/Bis gels.Proteins were then transferred onto Immobilon-PVDF membranes using thesame system. Membranes were prepared for immunoblotting by washing inTTBS (10 mM Tris-Glycine, pH 8.0, 0.15 M NaCl, with 0.05% (w/v)Tween-20). Membranes were then blocked in TTBS plus 5% (w/v) non-fat drymilk. The membranes were separated into high (>15 kD) and low MW (<15kd) ranges. Membranes were probed for specific proteins by overnightincubation with either a 1:1,000 dilution of rabbit anti-VMAT2 primaryantibody (Chemicon International, Temecula, Calif.) or a 1:400 dilutionanti-insulin primary antibody (Abcam, Cambridge, Mass.). The membraneswere then washed three times in TTBS and developed with 1:5,000 dilutionof either donkey anti-rabbit antibodies or sheep anti-mouse antibodiesconjugated to horseradish peroxidase (Amersham Bioscience, Pittsburgh,Pa.). After one hour, the membranes were washed in TTBS, and achemiluminescent substrate solution was added (Immobilon WesternSolution (Millpore, Bedford, Mass.)). Membranes were then used to exposeBio-Max film (Eastman Kodak, Rochester, N.Y.).

Statistical Analysis

All results are presented as means±S.E.M., or as indicated in the text.Students T testing was performed for assessing statistical significanceof differences. All p values are 2-tailed.

PET Study Protocol:

PET scans were performed on 12-14-week-old Lewis male rats. Prior toimaging, the animals were anesthetized by isoflurane inhalation. After atransmission scan of the area of interest had been acquired (used toperform attenuation correction of the emission data), the radioligand[¹¹C]DTBZ was administered (0.5-1.0 μCi/gm) in saline as a bolusinjection via the penile vein. PET scans of the animals were acquireddynamically to 90 minutes post-injection on a Concorde microPET-R4 (CTIMolecular Imaging (Knoxville, Tenn.)). The scanner provided a 100×80 mmfield of view with a reconstructed resolution of 2.25 mm in the central40 mm of the field of view.

At thirty minutes post injection of [¹¹C]DTBZ, animals received a secondinjection via the penile vein of cold analog. PET data were processedusing an attenuation correction matrix obtained by transmission scansand images were reconstructed using Fourier rebinning, followed bytwo-dimensional, filtered back projection using microPET managersoftware (CTI Molecular Imaging (Knoxville, Tenn.)).

Example 2 Glucose Tolerance in Adult Lewis Rats is Improved by TBZ

Older heavier Lewis rats display glucose intolerance relative to youngeranimals during an IPGTT (Natalucci, et al. 2003; Wang, et al. 1997). Toexplore the role of VMAT2 in insulin secretion, and to betterdemonstrate the possible value of VMAT2 as a potential therapeutictarget, older male Lewis rats (300-500 grams, >11 weeks of age) wereselected for IPGTT testing. For this study only rats with vehicle-aloneAUC IPGTT values greater than 10 g/dL×min were used. Doses oftetrabenazine (TBZ) were selected that were approximately three to tenfold higher than the equivalent doses currently used in humans to treatmovement disorders (Kenney and Jankovic 2006). Following TBZadministration (about 1 hour), but before glucose challenge, noreproducible differences were found in the baseline fasting glucoseconcentrations of control animals (data not shown). Following TBZtreatment and glucose challenge, however, a significant change was foundin the shape of the glucose disposition curve during IPGTT (FIG. 1A).

Comparison of the areas under the curve during IPGTT revealed that TBZreduced the glucose excursion by approximately 35% at 2.25 μg/gm bodyweight (FIG. 1A). This dose represented a maxima of the glucosetolerance enhancing effects of TBZ; at doses lower than 0.3 mg/Kg, theeffects of TBZ became undetectable by this assay, and at doses higherthan 5.0 mg/Kg, the AUC IPGTT became increasingly variable, oftensurpassing that of control levels. Chronic administration of TBZ atapproximately 0.1 mg/Kg body weight for five days suppressed the AUCIPGTT in a fashion similar to the single high dose (data not shown).

Example 3 TBZ Depletes Total Pancreatic Dopamine and L-DOPA ReversesEffects of TBZ

Dopamine is a well-known substrate of VMAT2-mediated vesicular transport(Howell, et al. 1994) and one of the main reported actions of TBZ is thedepletion of dopamine in brain tissue (Kenney and Jankovic 2006). Toexplore the possible role of dopamine in mediating the in vivo glucosetolerance enhancing effects of TBZ, the effects of TBZ on theconcentration of dopamine were examined in both the pancreas and thebrain. One hour after injection of TBZ, the dopamine content of bothtissues was significantly reduced (FIG. 2). Because islets compose onlyabout 2% of the pancreas, the marked effects of TBZ on total pancreaticdopamine content likely reflects dopamine depletion in non isletpancreatic tissue elements as well. The IPGTT experiments were repeatedwith TBZ. In these experiments, however, L-DOPA, the metabolic precursorof dopamine or a vehicle control, was administered one hour followingTBZ and concurrent with glucose.

The results revealed that L-DOPA (6.0 mg/Kg, via I.P. injection) wasable to reverse the effects of TBZ, increasing the AUC IPGTT to slightlybelow control levels.

Example 4 TBZ Enhances In Vivo Glucose-Dependent Insulin Secretion

The hypothesis that the smaller glucose excursions in IPGTT seen afteradministration of TBZ were due to increased insulin concentrations inthe plasma following glucose stimulation was tested. Blood glucose,plasma insulin and glucagon concentrations in blood samples obtainedduring IPGTT were measured (FIG. 3A-3D). Both area under the curve (AUC)insulin and glucagon (GCG) measurements were changed by administrationof TBZ. Plasma insulin amounts were significantly greater following asingle dose of TBZ or chronic low doses of TBZ (0.1 mg/Kg bodyweight/day×5 days) and glucose challenge relative to the vehicle-treatedcontrols. In addition, dopamine, given via i.v. injection at the sametime as glucose, partially blocked the insulin enhancing effects of TBZ(FIG. 3A). The AUC plasma glucagon measurements were lower relative tocontrols following i.v. TBZ administration and glucose challenge (FIGS.3B and 3D). The change in AUC glucagon however was less than the changein AUC insulin. In STZ treated rats which maintained glucose dependantinsulin secretion, TBZ (1.5 mg/Kg) increased the AUC insulin measurementby approximately 50%-80% and decreased AUC IPGTT (data not shown).

Example 5 TBZ Enhances In Vitro Glucose-Dependent Insulin Secretion inPurified Rat Islets

Because VMAT2 is located throughout the CNS and glucose homeostasis isregulated by the autonomic nervous system, a critical question waswhether TBZ was acting locally in islets. Whether the VMAT2 antagonistdihydrotetrabenazine (DTBZ), the direct and active metabolite of TBZ,could enhance insulin secretion in purified rat islets tested in vivowas tested. The islets were incubated in high and low glucose media withand without DTBZ. Insulin secretion increased ten fold in response toglucose, and was significantly further enhanced by DTBZ two to threefold (p<0.05) (FIG. 4). At low glucose, an increase in insulin secretionmediated by DTBZ was not statistically significant.

Example 6 DTBZ does not Act Through DPP-IV Inhibition

DTBZ structurally belongs to a class of quinolizine alkaloids. Recently,certain quinolizine alkaloids have been shown to increase insulin levelsthrough inhibiting dipeptidyl peptidase VI (DDP-IV), a serine proteasethat cleaves the insulin-stimulating incretin hormone glucagon-likepeptide-1 (Lubbers et al, 2007). To examine whether DPP-IV plays a rolein DTBZ's insulin enhancement, DTBZ's effect on DDP-IV in vitro wastested. DTBZ had no effect on DPP-IV at concentrations up to 10 μM (datanot shown).

Example 7 VMA T2 is Expressed in Rodent Islets and Beta Cells

As opposed to VMAT2 expressed by human beta cells (Anlauf et al. 2003),the presence of VMAT2 in rodent islets cannot be detected byimmunohistochemistry using currently available commercial antisera. Todemonstrate that VMAT2 is associated with rat islets, the followingseries of experiments were performed. First, total RNA was prepared frombrain, purified islets obtained from rodent pancreata, and totalpancreas. Total RNA was then reverse transcribed and amplified withspecific primers for rat VMAT2. A 175 bp cDNA fragment of the length andstructure expected from the published sequence of rat VMAT2 (Erickson,et al. 1992) was amplified and sequenced (FIG. 5A). Total RNA from brainwas used as a positive control (FIG. 5A, lane 1).

Quantitation of specific VMAT2 transcripts in islets total RNA versuscomplete pancreas total RNA showed that VMAT2-specific RNA wasenriched >10 fold in islets relative to total pancreas (FIG. 5A, lane 2versus lane 3). In the absence of the reverse transcription reaction, noPCR product was found (FIG. 5A, lane 4). Within the pancreas, insulinproducing beta cells uniquely express the GLUT2 transporter. The toxinSTZ selectively targets and destroys beta cells following transport byGLUT2 (Szkudelski 2001; Elsner, et al. 2000). To demonstrate that VMAT2is associated with beta cells of the endocrine pancreas, the selectivebeta cell toxicity of STZ was used. Total RNA from pancreata obtainedfrom four control rats and four STZ-induced diabetic rats was prepared.Quantitation of VMAT2 message by real time PCR showed that treatmentwith STZ significantly reduced the amount of VMAT2 in diabetic pancreatarelative to control pancreata 84 to 92% (99.9% Cl) (FIG. 5B). Whenprotein lysates were prepared from pancreata obtained from control ratsand STZ-induced diabetic rats, separated by SDS-PAGE, transferred tomembranes and then probed with VMAT2 antibodies, the loss of VMAT2protein, as well as preproinsulin protein, following STZ treatment wasvisible by the loss of the western blotting signal (FIG. 5C).

Example 8 Model for the Role of VMAT2 in Islet Function

Several previous studies have demonstrated a link between insulinsecretion and dopamine. For example, treatment of Parkinson's patientswith dopamine precursor, L-DOPA, reduces insulin secretion in glucosetolerance tests (Rosati, et al. 1976). In rodent experiments, i.v.administration of L-DOPA inhibits glucose-stimulated insulin secretion(Ericson, et al. 1977; Zern, et al. 1980). In culture, analogues ofdopamine inhibit glucose-stimulated insulin release by purified islets(Arneric, et al. 1984). Most recently, Rubi et al. (Rubi et al. 2005)demonstrated that mouse beta cells (INS-1E cells), as well as purifiedrat and human islets, express the dopamine D2 receptor. In these cellsand tissues, the D2 receptor was shown to colocalize with insulin insecretory granules in a pattern similar to the colocalization of VMAT2and insulin (Anlauf et al. 2003). Both dopamine and the D2-like receptoragonist, quinpirole, inhibited glucose-stimulated insulin secretion whentested in primary rat beta cells, and rat, mouse and human pancreaticislets.

Together with the studies of Rubi et al. (Rubi et al. 2005) and others(Brodoff and Kagan 1972) the following model for the role of VMAT2 inislet function can be proposed. Dopamine produced locally in the betacell cytoplasm is normally transported and stored in insulin-containingvesicles. In the presence of TBZ, the vesicular storage of dopaminedeclines. Under normal glucose stimulated insulin secretion, dopamine isco-released with insulin and acts either in an autocrine or paracrinefashion to limit glucose-stimulated insulin secretion by other localbeta cells. In the presence of TBZ, this negative feedback loop is notpresent and less dopamine is released with insulin and other local betacells remain uninhibited by dopamine. The studies disclosed hereinfocused on dopamine as the most likely intermediate mediator of theeffects of TBZ, although it is not ruled out that other monoamines, suchas serotonin, etc., also play a role in the observed in vivo effects ofTBZ.

Example 9 Repeated Low Doses of TBZ May Also be Active in ReducingGlucose Excursions

Currently, arginine pulse stimulation of insulin secretion is a goldstandard measurement for evaluating functional beta cell mass.Preliminary studies by the inventors with TBZ suggest that more detailedglucose clamp and insulin secretion measurements should be performed,and the inventors continue to evaluate whether inhibition of VMAT2 mightfurther improve the hyperglycemic clamp technique applied to evaluatingbeta cell mass. To date, the inventors have found that repeated lowdoses of TBZ may also be active in reducing glucose excursions. In otherstudies the inventors have found some evidence from PET pharmacokineticstudies that DTBZ may accumulate in the pancreas. Together these datasuggest that chronic low doses of TBZ may also result in antagonism ofVMAT2.

Example 10 Synthesis of Novel VMAT2 Antagonists

In an effort to generate novel VMAT2 antagonists, compound 1 (shown inFIG. 6), a simplified analog of DTBZ, was synthesized. As shown in FIG.9, veratraldehyde (compound 2) was treated with ammonium acetate at 50°C. for 3 hours and converted into β-amino acid (compound 3) bycondensation with malonic acid at 80° C. for 24 hours. Protection withBoc anhydride at room temperature for 24 hours and subsequentcondensation with potassium malonate methyl ester for 2 days at roomtemperature led to a β-keto ester (compound 4). Alkylation with isobutylbromide in the presence of potassium carbonate for 3 hours at refluxtemperature afforded a mixture of compound 5 and compound 6 in moderateyield.

After removal of the Boc group, the mixture was treated with sodiumbicarbonate in methanol for 24 hours at room temperature to yield thecyclized products, compound 7 and compound 8 quantitatively. Compound 9was obtained from reduction of compound 7 with sodium borohydride atroom temperature for 1 hour and then further converted to compound 1 andits diastereoisomers with lithium aluminum hydride.

Example 11 Glucose Tolerance Tests of Novel Compounds

Racemic compound 1 and its diastereoisomers were evaluated for theirability to improve glucose tolerance by IPGTTs in rats. These newanalogs were less potent than TBZ possibly due to diminished affinityfor VMAT2 (FIG. 7). Accordingly, these analogs were not further pursued.

During random screens of intermediates generated in the course of thesynthesis of compound 1, it was found that compound 8, a noveldihydropyridone resulted from the competing O- versus C-alkylation ofenolic β-keto ester compound 4 followed by cyclization, showed potenthypoglycemic effect. As illustrated in FIG. 7, compound 8 decreased theAUC IPGTT by 45% at the dose of 2 mg/kg compared to 26% for TBZ.

Example 12 Synthesis and Characterization of Analogs of Compound 8

Prompted by this surprising result, analogs of compound 8 were designedand synthesized.

As outlined in FIG. 10, veratraldehyde (compound 2) was first condensedwith ethyl acetoacetate at −5° C. for 1.5 hours, and spontaneouscyclization yielded lactone, compound 10. Using potassium carbonate asthe base, O-alkylation of compound 10 with methyl bromide or isobutylbromide provided compound 11 and compound 12.

Similarly, compounds 15 and 16 were prepared from dihydroisoquinoline,compound 13, via condensation with dimethyl 1,3-acetonedicarboxylatefollowed by cyclization and alkylation.

Aromatization induced by 2,3-dichloro-5,6-dicyanobenzoquinone (DDQ) andacidic hydrolysis of compound 8 afforded compound 17 and compound 18,respectively. Using conventional methods, other analogs of compound 8may be readily synthesized and tested for activity using, e.g., themethods disclosed herein.

Analogs prepared as above were tested for their hypoglycemic activitiesin rats using the IPGTT protocol. Interestingly, the hypoglycemiceffects of these compounds were only seen following glucose stimulation.Results shown in FIG. 7 demonstrated that the dihydropyridone scaffoldin compound 8 is essential for the hypoglycemic activity. Replacementwith dihydropyrone (compound 11 and compound 12), oxidation, orhydrolysis of 8 (compound 17, compound 18) resulted in the total loss ofactivities.

Interestingly, the rigid derivatives, compound 15 and compound 16 wereactive but less potent, in opposition to the structure-activityrelationship trend seen in compound 1 and DTBZ.

Analytical data for compound 8 are as follows: ¹H NMR (300 MHz, CDCl₃) δ6.90-6.85 (m, 2H), 6.84 (s, 1H), 5.45 (br, 1H), 5.07 (s, 1H), 4.69-4.63(dd, 1H), 3.87 (s, 3H), 3.86 (s, 3H), 3.64-3.60 (m, 2H), 2.71-2.47 (m,2H), 2.02-1.96 (m, 1H), 0.96-0.93 (dd, 6H); ¹³C NMR (75 MHz, CD₃OD) δ170.0, 168.9, 149.4, 149.1, 133.5, 119.0, 111.4, 109.3, 93.8, 75.1,56.3, 56.2, 55.0, 37.3, 28.1, 19.5, 19.4; ESI-MS (M⁺+H): 306.3.

Analytical data for compound 15 are as follows: ¹H NMR (300 MHz, CDCl₃)δ 6.58 (s, 1H), 6.54 (s, 1H), 5.15 (s, 1H), 4.71-4.65 (m, 2H), 3.80 (s,3H), 3.79 (s, 3H), 3.67 (s, 3H), 2.81-2.60 (m, 4H), 2.48-2.37 (m, 1H);¹³C NMR (75 MHz, CD₃OD) δ 168.3, 167.4, 148.0 (2C), 127.4, 127.3, 111.3,108.6, 94.6, 56.3, 56.2, 56.0, 54.1, 38.4, 37.2, 29.4; ESI-MS (M⁺+H):290.1.

Example 13 Compound 8 Binds to VMAT2

To test whether the strong hypoglycemic effect of compound 8 isconferred by binding to VMAT2 in beta cells, a PET study in rats wasperformed. The animals were treated with radiolabeled DTBZ, and theuptake of [¹¹C]DTBZ in pancreas was monitored by PET scan in thepresence of an excess of compound 8.

Contrary to the inventors' original interpretation (Xie et al., 2008),it is now believed that compound 8 does show binding to VMAT2. Acomparison of the binding curves for the displacement of [¹¹C]DTBZ inthe endocrine pancreas by compound 8 (FIG. 8) versus the displacement of[¹¹C]DTBZ by authentic cold DTBZ (FIG. 9) suggests that there is atleast a weak binding of compound 8 to VMAT2 relative to DTBZ.Alternatively, VMAT2 is known to possess multiple binding sites(Darchen, et al., 1989 and Scherman et al., 1984) and one couldhypothesize that compound 8 is binding to a site dissimilar to that usedby DTBZ.

An alternative mechanism of action, suggested by the inventors' PETstudies and in vivo experiments might be that compound 8 is acompetitive inhibitor of DA transport with no D2R action or is acompetitive inhibitor of DA transport with D2R antagonist activity. Ithas been demonstrated that [¹¹C](+)DTBZ binding to VMAT2 is sensitive tochanges in vesicular DA storage levels (Tong et al., 2008). If compound8 is transported by VMAT2 into vesicles, at the expense of DA, thiscould alter [¹¹C](+)DTBZ binding to VMAT2.

A high throughput (HTP) assay was developed to screen for compounds thatinteract with, e.g., bind to, VMAT2. The HTP exploits a class ofcompounds called Fake Fluorescent Neurotransmitters (FFN) that share thefollowing characteristics a) low molecular weight and sufficientlyhydrophilic to be water soluble; b) in aqueous buffer (pH 7), theexcitation maxima is around 380-406 nm and the emission maxima is around501 nm; and c) similar Kd for VMAT2 as dopamine. To validate the use ofthese compounds in beta cell lines (positive for VMAT2) the followingset of experiments were performed.

The rat INS 832/13 cell line was selected because it secretes insulin inresponse to glucose concentrations in the physiological range and can beshown to contain VMAT2 by PCR. INS 832/13 cells were stably transfectedwith a plasmid containing the human proinsulin gene and after subcloningwas found to be responsive to glucose in terms of insulin secretion(Hohmeier et al., 2000). INS cells were grown on cover slips in normalmedia, then pretreated with and without DTBZ in low glucose media forone hour and then loaded with FFN (GH206). The distribution offluorescent dye was then examined under a fluorescent microscope. TheFFN distributes itself intracellularly in a punctate pattern similar, ifnot identical, to insulin containing granules of this cell line (datanot shown). In addition, it was observed that DTBZ exhibited a blockeffect on the uptake of FFN (data not shown). For better quantitation ofthe fluorescent signal, the cells were grown in 46 well plates and thesame experiment substantially repeated. Briefly, cells were grown toconfluence in complete high glucose media, then incubated for one hourin low glucose media. DTBZ was then added to the wells for one hour,followed with a second one hour incubation with FFN. Wells were thenwashed and the fluorescent signal (380 exc, 512 emm) measured in amultiwell fluorometer (Synergy HT Multi-Mode Microplate Reader).

The measurements of fluorescence (data not shown) confirmed that DTBZwas able to block uptake of FFN in these cells and suggested that thisassay format may be suitable for detecting other compounds that act ascompetitive inhibitors of DA uptake by VMAT2 as well as antagonists ofVMAT2 DA transport.

To test whether this assay was suitable to screen synthetic analogs ofTBZ for inhibition of VMAT2 function, compounds 8, 11, and 18 weretested. The results of that assay are shown in FIG. 10. These resultssuggest that compound 8 is able to block FFN transport into INSvesicles, although its activity on a per mole basis seems less than thatof DTBZ.

Example 13 Compound 8 does not Act Through DPP-IV Inhibition

DTBZ and its analogs are structurally similar to a class of quinolizinealkaloids previously shown to inhibit dipeptidyl peptidase IV (DPP-IV)(Lubbers et al, 2007), but compound 8 tested negative against DPP-IV invitro at concentrations up to 10 μM (data not shown). Therefore, themechanism underlying the anti-hyperglycemic effect of compound 8 is notthrough DPP-IV inhibition.

Pending U.S. Provisional Application Ser. No. 61/188,419 filed Aug. 8,2008 entitled Hypoglycemic Dihydropyridones, International ApplicationNo. PCT/US08/03338 entitled Methods and Compositions for ModulatingInsulin Secretion and Glucose Metabolism, which was filed on Mar. 12,2008, U.S. Provisional Application Ser. No. 60/906,623, filed on Mar.12, 2007, and U.S. Provisional Application Ser. No. 60/932,810, filedMay 31, 2007, are incorporated herein by reference in their entiretiesfor all purposes.

The present invention is not to be limited in scope by the specificembodiments described herein. Indeed, various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description and theaccompanying figures. Such modifications are intended to fall within thescope of the appended claims.

CITED DOCUMENTS

The following documents, including all documents cited above, areincorporated by reference as if recited in full herein:

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1. A compound of formula I:

wherein R₁, R₂, R₃, R₆, and R₇ are independently selected from the groupconsisting of H, halogen, hydroxyl, C₁₋₈alkyl, C₁₋₈alkenyl, C₁₋₈alkynyl,C₁₋₈alkoxy, 3- to 8-membered carbocyclic or heterocyclic, aryl,heteroaryl, C₁₋₄aralkyl, residues of glycolic acid, ethyleneglycol/propylene glycol copolymers, carboxylate, ester, amide,carbohydrate, amino acid, alditol, OC(X)₂COOH, SC(X)₂COOH, NHCHXCOOH,COY, CO₂Y, sulfate, sulfonamide, sulfoxide, sulfonate, sulfone,thioalkyl, thioester, propylphthalimide, and thioether; R₄ and R₅, whichare attached to one or more positions of at least one carbon atom of therespective rings, are independently selected from the group consistingof H, halogen, hydroxyl, C₁₋₈alkyl, C₁₋₈alkenyl, C₁₋₈alkynyl,C₁₋₈alkoxy, 3- to 8-membered carbocyclic or heterocyclic, aryl,heteroaryl, C₁₋₄aralkyl, residues of glycolic acid, ethyleneglycol/propylene glycol copolymers, carboxylate, ester, amide,carbohydrate, amino acid, alditol, OC(X)₂COOH, SC(X)₂COOH, NHCHXCOOH,COY, CO₂Y, sulfate, sulfonamide, sulfoxide, sulfonate, sulfone,thioalkyl, thioester, propylphthalimide, and thioether; X is selectedfrom the group consisting of H, C₁₋₈alkyl, C₁₋₈alkenyl, C₁₋₈alkynyl,carbocycle, aryl, heteroaryl, heterocycle, alkylaryl, alkylheteroaryl,and alkylheterocycle, wherein each alkyl, carbocycle, aryl, heteroaryl,heterocycle, alkylaryl, alkylheteroaryl, and alkylheterocycle isoptionally substituted with at least one substituent; Y is selected fromthe group consisting of H, C₁₋₈alkyl, C₁₋₈alkenyl, C₁₋₈alkynyl, aryl,carbocycle, heteroaryl, heterocycle, alkylaryl, alkylheteroaryl,alkylheterocycle, and heteroaromatic, wherein each alkyl, alkenyl,alkynyl, aryl, carbocycle, heteroaryl, heterocycle, alkylaryl,alkylheteroaryl, alkylheterocycle, and heteroaromatic is optionallysubstituted with at least one substituent; and --- is an optional bond,wherein the optional bond is a single bond or a double bond; or anenantiomer, optical isomer, diastereomer, N-oxide, crystalline form,hydrate, or pharmaceutically acceptable salt thereof.
 2. The compoundaccording to claim 1, wherein the compound has formula II:

wherein R₁, R₂, R₃, R₈ and R₉ are independently selected from the groupconsisting of H, halogen, C₁-C₈ alkyl, C₁-C₈alkenyl, and C₁-C₈alkynyl;R₄ and R₅, which are attached to one or more positions of at least onecarbon atom of the respective rings, are independently selected from thegroup consisting of H, halogen, hydroxyl, C₁₋₈alkyl, C₁₋₈alkenyl,C₁₋₈alkynyl, and C₁₋₈alkoxy; and --- is an optional bond, wherein theoptional bond is a single bond or a double bond; or an enantiomer,optical isomer, diastereomer, N-oxide, crystalline form, hydrate, orpharmaceutically acceptable salt thereof.
 3. The compound according toclaim 2, wherein the compound has formula III:

wherein R₁, R₂, and R₃ are independently selected from the groupconsisting of H, C₁-C₈alkyl, C₁-C₈alkenyl, and C₁-C₈alkynyl; and --- isan optional bond, wherein the optional bond is a single bond or a doublebond; or an enantiomer, optical isomer, diastereomer, N-oxide,crystalline form, hydrate, or pharmaceutically acceptable salt thereof.4. The compound according to claim 3, wherein R₁ is selected from thegroup consisting of H, C₁-C₈alkyl, C₁-C₈alkenyl, and C₁-C₈alkynyl; R₂and R₃ are selected from the group consisting of H and C₁alkyl; and ---is a single bond if R₂ or R₃ is not H.
 5. The compound according toclaim 4, wherein the compound is compound 15:

or an enantiomer, optical isomer, diastereomer, N-oxide, crystallineform, hydrate, or pharmaceutically acceptable salt thereof.
 6. Thecompound according to claim 1, wherein the compound has formula IV:

wherein R₁, R₈, and R₉ are independently selected from the groupconsisting H, halogen, hydroxyl, C₁₋₈alkyl, C₁₋₈alkenyl, C₁₋₈alkynyl,C₁₋₈alkoxy, 3- to 8-membered carbocyclic or heterocyclic, aryl,heteroaryl, C₁₋₄aralkyl, residues of glycolic acid, ethyleneglycol/propylene glycol copolymers, carboxylate, ester, amide,carbohydrate, amino acid, alditol, OC(X)₂COOH, SC(X)₂COOH, NHCHXCOOH,COY, CO₂Y, sulfate, sulfonamide, sulfoxide, sulfonate, sulfone,thioalkyl, thioester, propylphthalimide, and thioether; X is selectedfrom the group consisting of H, C₁₋₈alkyl, C₁₋₈alkenyl, C₁₋₈alkynyl,carbocycle, aryl, heteroaryl, heterocycle, alkylaryl, alkylheteroaryl,and alkylheterocycle, wherein each alkyl, carbocycle, aryl, heteroaryl,heterocycle, alkylaryl, alkylheteroaryl, and alkylheterocycle isoptionally substituted with at least one substituent; Y is selected fromthe group consisting of H, C₁₋₈alkyl, C₁₋₈alkenyl, C₁₋₈alkynyl, aryl,carbocycle, heteroaryl, heterocycle, alkylaryl, alkylheteroaryl,alkylheterocycle, and heteroaromatic, wherein each alkyl, alkenyl,alkynyl, aryl, carbocycle, heteroaryl, heterocycle, alkylaryl,alkylheteroaryl, alkylheterocycle, and heteroaromatic is optionallysubstituted with at least one substituent; or an enantiomer, opticalisomer, diastereomer, N-oxide, crystalline form, hydrate, orpharmaceutically acceptable salt thereof.
 7. The compound according toclaim 6, wherein R₁ is —CH₂—CH—(CH₃)₂.
 8. The compound according toclaim 6, wherein R₈ is methyl.
 9. The compound according to claim 6,wherein R₉ is methyl.
 10. The compound according to claim 6, whereinboth R₈ and R₉ are methyl.
 11. The compound according to claim 6,wherein R₁ is selected from the group consisting of C₁₋₈alkyl,C₁₋₈alkenyl, C₁₋₈alkynyl, and C₁₋₈alkoxy and R₈ and R₉ are both methyl.12. The compound according to claim 6, which is compound 8:

or an enantiomer, optical isomer, diastereomer, N-oxide, crystallineform, hydrate, or pharmaceutically acceptable salt thereof.
 13. Acompound having the structure (1):

or an enantiomer, optical isomer, diastereomer, N-oxide, crystallineform, hydrate, or pharmaceutically acceptable salt thereof.
 14. Apharmaceutical composition comprising a pharmaceutically acceptablecarrier and a therapeutically effective amount of a compound accordingto claim
 1. 15. A method for modulating blood glucose levels in asubject comprising administering to a subject an effective amount of thecompound according to claim
 1. 16. A method for preventing, treating, orameliorating the effects of diabetes in a subject comprisingadministering to a subject an effective amount of the compound accordingto claim
 1. 17. A method for preventing, treating, or ameliorating theeffects of hyperglycemia comprising administering to a subject aneffective amount of the compound according to claim
 1. 18. A method formodulating blood glucose levels in a subject comprising administering toa subject an effective amount of the pharmaceutical compositionaccording to claim
 14. 19. A method for preventing, treating, orameliorating the effects of diabetes in a subject comprisingadministering to a subject an effective amount of the pharmaceuticalcomposition according to claim
 14. 20. A method for preventing,treating, or ameliorating the effects of hyperglycemia comprisingadministering to a subject an effective amount of the pharmaceuticalcomposition according to claim
 14. 21. A method for modulating bloodglucose levels in a subject comprising administering to a subject aneffective amount of the compound according to claim 1 or apharmaceutical composition thereof, which compound or compositioninteracts with VMAT2 to provide the modulation.
 22. A method forpreventing, treating, or ameliorating the effects of diabetes in asubject comprising administering to a subject an effective amount of thecompound according to claim 1 or a pharmaceutical composition thereof,which compound or composition interacts with VMAT2 to provide theprevention, treatment, or amelioration of the effects of diabetes in thesubject.
 23. A method for preventing, treating, or ameliorating theeffects of hyperglycemia comprising administering to a subject aneffective amount of the compound according to claim 1 or apharmaceutical composition thereof, which compound or compositioninteracts with VMAT2 to provide the prevention, treatment, oramelioration of the effects of hyperglycemia in the subject.